CN110358535A - The fluorescent carbon point nano-probe and its application method of hydrogen sulfide imaging in hydrogen sulfide and living cells are detected based on inner filtering effect - Google Patents
The fluorescent carbon point nano-probe and its application method of hydrogen sulfide imaging in hydrogen sulfide and living cells are detected based on inner filtering effect Download PDFInfo
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Abstract
The present invention proposes fluorescent carbon point nano-probe and its application method that hydrogen sulfide imaging in hydrogen sulfide and living cells is detected based on inner filtering effect, establishes the simple green method of a kind of fluorescence detection hydrogen sulfide and living cells hydrogen sulfide tracer.Fluorescent carbon point is prepared for by raw material of tomato, the carbon dots (CDs) are under 410 nm ultraviolet lights, very strong green fluorescence is generated at 520 nm, fluorescence emission peak gradually red shift with the increase of excitation wavelength, relative fluorescence quantum yield is 53%, and inner filtering effect can be based on as fluorogenic donor and small organic molecule receptor (DMI) and constitute probe, detection for external source and Endogenous Hydrogen Sulfide, the sensor-based system is as fluorescence probe, it has been successfully applied to detection hydrogen sulfide and living cells imaging analysis, the method for sensing is in environmental analysis field, it has broad application prospects in biochemical analysis field and cell imaging analysis.
Description
Technical field
The present invention relates to biochemical analysis technical field, particularly relate to based on sulphur in inner filtering effect detection hydrogen sulfide and living cells
Change the fluorescent carbon point nano-probe and its application method of hydrogen imaging.
Background technique
Carbon quantum dot (CDs), also known as carbon dots or carbon nano dot, are a kind of novel carbon nanomaterials, and size generally exists
10nm hereinafter, have excellent optical and electrical properties, particle uniformly, good dispersion and low preparation cost, low toxicity, good life
Object compatibility, excitation and the features such as launch wavelength is adjustable, good light stability, unglazed scintillation.CDs is compared and traditional metal
Quantum dot has many advantages, and unique luminosity and biocompatibility are in luminaire, photocatalysis, biochemical analysis, photoelectricity
The fields such as son, cell imaging and detection are with good application prospect.
The preparation of CDs has had made great progress, at present it has been reported that the method for many synthesis CDs, mainly has: electric arc
Electric discharge (X. Y. Xu, R. Ray, W. A. Scrivens, J. Am. Chem. Soc. 2004,126,12736-
12737), laser ablation method (X. Wang, L. Cao, Y. Sun, Chem. Comrmrn, 2009,3774-
3776), electrochemical process (H. Li, X. He, Z. Kang, Angew. Chem. Int. Ed, 2010,49,4430-
4434), combustion method (H. Liu, T. Ye, C. Mao, Angew, Chem. Int. Ed. 2007,46,6473-
6475), hydro-thermal method (M. X. Gao, C. F. Liu, Z. L. Wu, Q. L. Zeng, X. X. Yang, W. B.
Wu, Y. F. Li and C.Z. Huang, Chem. Commun., 2013,49,8015-8017), Ultrasonic treatment
(H.T. Li, X.D. He, Z.H. Kang, Carbon, 2011,49,605-609), microwave method (J. L. Chen,
X. P. Yan, Chem. Commun, 2011,47,3135-3137) etc..For this method, some raw material valuableness are not
It is easy to receive, some process complexity are cumbersome, and some reaction conditions are harsher, and energy consumption is bigger.Therefore, it finds a kind of green
Colour circle guarantor, low energy consumption, low cost synthesis CDs be very urgent and significant.
Hydrogen sulfide has weight as the third intracorporal endogenous gas signaling molecule in various physiology and pathologic process
It acts on, especially the hydrogen sulfide characteristic with very strong endogenous anti-oxidative effect and neuroprotection, is important interior of one kind
The gasotransmitter of natural disposition facilitates diversified biochemical reaction process, such as: blood vessel dilatation, revascularization, Apoptosis,
Inflammation and nerve modulation etc..A kind of fluorescence enhancement type sulphur based on chromocor derivative is disclosed in patent CN201310381224.7
Change hydrogen fluorescence probe, it is good to provide a kind of selectivity, high sensitivity, can fluorescence enhancement detect hydrogen sulfide containing fluorescence probe.
The fluorescence probe is only for the presence or absence of detection hydrogen sulfide；Patent CN201210348010.5 is related to for detecting hydrogen sulfide
(H2S fluorescence probe), specifically a kind of fluorescence probe and its application that detection intracellular hydrogen sulfide is restored based on nitro.
Fluorescence probe is and the introducing meta nitro aniline or m-nitro on parent using hemicyanine dye or cyanine dye as fluorescent parent
Phenol.Detection of the fluorescence probe for hydrogen sulfide inside and outside water body or organism.Existing fluorescence probe is only capable of detecting to be measured
Whether contain hydrogen sulfide in sample, and can not continuously detect the content of hydrogen sulfide.
Summary of the invention
The present invention proposes that the fluorescent carbon point nanometer that hydrogen sulfide imaging in hydrogen sulfide and living cells is detected based on inner filtering effect is visited
Needle and its application method establish the simple green method of a kind of fluorescence detection hydrogen sulfide and living cells hydrogen sulfide tracer.
The technical scheme of the present invention is realized as follows:
Detect the fluorescent carbon point nano-probe of hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect, fluorescent carbon point with have
Machine small molecule receptor is based on inner filtering effect and forms fluorescent carbon point nano-probe.
The fluorescent carbon point is prepared by tomato carbonization, and small organic molecule receptor chemistry formula is。
The synthetic method of the fluorescent carbon point, steps are as follows: new fresh tomato being placed in mortar, mistake after being fully ground
100-260 mesh filter membrane obtains filtrate, takes the filtrate of certain volume in hydrothermal reaction kettle, adds ultrapure water and is heated to 250 DEG C simultaneously
It stirs, stops heating cooled to room temperature after 10h, filtering is collected filtrate and is placed in bag filter, is protected from light under ultrapure water environment
For 24 hours, taking supernatant is fluorescent carbon point for dialysis.
The volume ratio of the filtrate and ultrapure water is 3:(20-23)；Bag filter model MWCO:1 kDa, pore
size: ca. 1.0 nm。
The synthetic method of the small organic molecule receptor, steps are as follows:
(1) take 2,3,3- trimethyl -3H- indoles and iodomethane mixed dissolution in acetonitrile, under protection of argon gas 60 DEG C of reflux
11h naturally cools to room temperature, obtains a kind of lightpink precipitating, and filtering is washed three times with ethyl acetate, and vacuum drying obtains lightpink
Powder；Synthetic route are as follows:；
(2) lightpink powder and 4- diaminobenzene formaldehyde (0.112g, 0.75mmoL) mixed dissolution that step (1) obtains are existed
In 10mL ethyl alcohol, 180 DEG C of reflux 4.5h, are evaporated under reduced pressure, remaining liq obtains pure through column Chromatographic purification under protection of argon gas
Small organic molecule receptor, synthetic route are as follows:
。
Substance withdrawl syndrome of the 2,3,3- trimethyl -3H- indoles in acetonitrile is (1-4) mmol/ in the step (1)
The amount concentration of the tie substance of mL, iodomethane in acetonitrile is followed successively by (2-8) mmol/mL.
In the step (2), the substance withdrawl syndrome of lightpink powder in ethanol is 0.05mmol/mL, 4- diamino
The substance withdrawl syndrome of benzaldehyde in ethanol is 0.075mmol/mL；The reagent that column Chromatographic purification uses is methylene chloride and second
Alcohol is by volume (20-23): 1.
The application method of the fluorescent carbon point nano-probe, step are as follows: fluorescent carbon point and small organic molecule receptor exist
24 h are incubated for living cells in Tissue Culture Dish, the liquid that cell removing does not enter cell are washed with PBS buffer solution, using laser
Laser Scanning Confocal Microscope system is observed, and the sodium sulfide solution for being then separately added into multiple groups gradient concentration again is incubated for 1h, slow with PBS
Fliud flushing washing cell removes the liquid for not entering cell, is observed using laser confocal microscope system.
The invention has the following advantages:
(1) present invention by raw material of tomato is prepared for fluorescent carbon point, the carbon dots (CDs) under 410 nm ultraviolet lights,
Very strong green fluorescence, fluorescence emission peak gradually red shift, relative fluorescence with the increase of excitation wavelength are generated at 520 nm
Quantum yield is 53%(using quinine sulfate as reference), C, N, O constituent content are respectively 60.23%, 7.16%, 30.11%, and energy
Inner filtering effect is based on as fluorogenic donor and small organic molecule receptor (DMI) and constitutes probe, is used for external source and Endogenous Hydrogen Sulfide
Detection.
(2) fluorescent carbon point of the invention is incubated for 24 h with living cells in Tissue Culture Dish, micro- using laser co-focusing
Mirror system is observed, and cell continuously sends out green fluorescence, is added small organic molecule and is incubated for fluorescent quenching after 12 h, is being added
Fluorescence restores after external source hydrogen sulfide is incubated for 1h, is based on above-mentioned characteristic, establishes a kind of fluorescence detection hydrogen sulfide and living cells vulcanization
The simple green method of hydrogen tracer.
(3) present invention firstly discovers that by the carbon dots of tomato carbonization hydrogen sulfide, this method can be detected by inner filtering effect
As the effective means of detection hydrogen sulfide, significant advantage is shown.Detection is limited down to 0.42 μm of ol/L, linear response range
Are as follows: 10-70 μm of ol/L(R 2=0.9904), which has simple, low cost, green, high selection as fluorescence probe
Property, optical signature quickly, sensitive, be successfully applied to detection hydrogen sulfide and living cells imaging analysis, the method for sensing is in ring
It has broad application prospects in border analysis field, biochemical analysis field and cell imaging analysis.
(4) present invention use the raw material of carbon dots synthesis for tomato, cheap, source is wide for Henan area；It adopts
It is carbonized with pyrolysis way, has many advantages, such as that Preparation equipment instrument is simple, carbonization time is short, merchandized handling may be implemented；Have
The sensitivity of height and good selectivity, linear response range are wide；Based on inner filtering effect, with more simple and convenient, mechanism is bright
The unique advantages such as true；Fluorescence probe detection hydrogen sulfide rapid sensitive, the response time is short, specificity is good；The fluorescence probe living cells
Hydrogen sulfide imaging analysis is stablized, and green fluorescence is presented；Therefore, which can be used as fluorescence probe detection hydrogen sulfide and living cells
Interior hydrogen sulfide imaging analysis, due to its simple and convenient, inexpensive, environmentally protective, highly selective, quick, sensitive optical signature,
It has a good application prospect.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is the transmission electron microscope picture (a) and grain size distribution (b) of fluorescence CDs.
The ultraviolet-visible absorption figure (a) and fluorescence excitation figure (b) and transmitting (c) figure that Fig. 2 is fluorescence CDs.
The emission peak (a) and DMI ultraviolet-visible absorption overlap of peaks figure (b) and sodium sulfide solution is added that Fig. 3 is fluorescence CDs
Quench (c) afterwards.
Fig. 4 is quenching figure of the DMI to fluorescent carbon point of various concentration, wherein from top to bottom concentration is successively got higher.
Fig. 5 is the living cells image of fluorescent carbon point, probe and various concentration sodium sulfide solution and probe.
Fig. 6 is that the sodium sulfide solution of various concentration restores figure to the fluorescence of probe, wherein from top to bottom concentration is successively got higher.
Fig. 7 is that probe schemes the specificity of hydrogen sulfide.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described, it is clear that institute
The embodiment of description is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention,
Those of ordinary skill in the art's every other embodiment obtained under that premise of not paying creative labor, belongs to this hair
The range of bright protection.
Embodiment 1
The preparation method of the fluorescent carbon point nano-probe of hydrogen sulfide imaging in hydrogen sulfide and living cells is detected based on inner filtering effect:
1. the synthesis of fluorescence nano carbon dots
Fluorescence nano carbon dots (CDs) are made by pyrolysis tomato, specific as follows: being weighed new fresh tomato 5.0g in mortar, filled
Divide after grinding through 150 mesh membrane filtrations, takes filtrate 3mL in hydrothermal reaction kettle, add 20mL ultrapure water and be heated to 250 DEG C simultaneously
Stirring stops heating and naturally cools to room temperature, filters, take filtrate in bag filter (MWCO:1 kDa, pore after 10 hours
Size:ca. 1.0 nm) in be protected from light in the environment of ultrapure water dialysis for 24 hours, take supernatant liquor to be kept in dark place in 4 DEG C.Fig. 1 gives
The transmission electron microscope picture and grain size distribution of synthesized green carbon quantum dot are gone out, from Tu Ke get, quantum dot is uniformly dispersed, and partial size is about
For 3nm.The ultraviolet-visible absorption figure (a) and fluorescence that Fig. 2 is fluorescence CDs excite (b) and transmitting (c) figure, and excitation wavelength is
410nm, launch wavelength 520nm are in apparent green fluorescence.
2. the synthesis of DMI
2,3,3- trimethyl -3H- indoles (3.2g, 20mmoL) and iodomethane (5.68g, 40mmoL) mixed dissolution are in 10mL acetonitrile
In, 60 DEG C of reflux 11h, naturally cool to room temperature under protection of argon gas, obtain a kind of lightpink precipitating, and ethyl acetate is used in filtering
It washes three times, vacuum drying obtains lightpink powder (5.8g, 96%)
(lightpink powder)；
Above-mentioned lightpink powder (0.150g, 0.50mmoL) and 4- diaminobenzene formaldehyde (0.112g, 0.75mmoL) is taken to mix molten
Solution is in 10mL ethyl alcohol, and 180 DEG C of reflux 4.5h, are evaporated under reduced pressure, remaining liq is through column Chromatographic purification (dichloromethane under protection of argon gas
Alkane: ethyl alcohol=20:1 volume ratio) it obtains pure small organic molecule (DMI), it reacts as follows:
；
3. the building of the fluorescence probe based on inner filtering effect: the emission peak (a) and DMI ultraviolet-visible that Fig. 3 is fluorescence CDs are inhaled
It receives overlap of peaks figure (b) and quenches (c) after sodium sulfide solution is added.By ultraviolet-visible spectrum analysis, DMI has most at 546nm
Big absorption peak, simultaneously synthesizing CDs have maximum emission peak (λ at 520nmex=410nm), the two, which has, to be largely overlapped, i.e.,
DMI can largely sponge the transmitting light of CDs, show as CDs fluorescent quenching, and inner filtering effect occurs, successfully constructs fluorescence
Probe.As shown in Figure 4 and Figure 5, with the increase that DMI concentration is added, the fluorescence intensity of carbon quantum dot is constantly reduced, and is worked as and added
Fluorescence intensity is gradually recovered again after entering the hydrogen sulfide of various concentration, and centainly in a linear relationship.
Embodiment 2
1. the synthesis of fluorescence nano carbon dots
Fluorescence nano carbon dots (CDs) are made by pyrolysis tomato, specific as follows: being weighed new fresh tomato 5.0g in mortar, filled
Divide after grinding through 150 mesh membrane filtrations, takes filtrate 3mL in hydrothermal reaction kettle, add 21mL ultrapure water and be heated to 250 DEG C simultaneously
Stirring stops heating and naturally cools to room temperature, filters, take filtrate in bag filter (MWCO:1 kDa, pore after 10 hours
Size:ca. 1.0 nm) in be protected from light in the environment of ultrapure water dialysis for 24 hours, take supernatant liquor to be kept in dark place in 4 DEG C.
2. the synthesis of DMI
2,3,3- trimethyl -3H- indoles (1.6g, 10mmoL) and iodomethane (2.84g, 20mmoL) mixed dissolution are in 10mL acetonitrile
In, 60 DEG C of reflux 11h, naturally cool to room temperature under protection of argon gas, obtain a kind of lightpink precipitating, and ethyl acetate is used in filtering
It washes three times, vacuum drying obtains lightpink powder (2.9g, 96%)
(lightpink powder)；
Above-mentioned lightpink powder (0.150g, 0.50mmoL) and 4- diaminobenzene formaldehyde (0.112g, 0.75mmoL) is taken to mix molten
Solution is in 10mL ethyl alcohol, and 180 DEG C of reflux 4.5h, are evaporated under reduced pressure, remaining liq is through column Chromatographic purification (dichloromethane under protection of argon gas
Alkane: ethyl alcohol=20:1 volume ratio) it obtains pure small organic molecule (DMI), it reacts as follows:
；
3. the building of the fluorescence probe based on inner filtering effect: passing through ultraviolet-visible spectrum analysis, DMI has maximum suction at 546nm
Peak is received, simultaneously synthesizing CDs has maximum emission peak (λ at 520nmex=410nm), the two, which has, to be largely overlapped, i.e. DMI energy
The transmitting light for largely sponging CDs shows as CDs fluorescent quenching, and inner filtering effect occurs, successfully constructs fluorescence probe.
Embodiment 3
1. the synthesis of fluorescence nano carbon dots
Fluorescence nano carbon dots (CDs) are made by pyrolysis tomato, specific as follows: being weighed new fresh tomato 5.0g in mortar, filled
Divide after grinding through 150 mesh membrane filtrations, takes filtrate 3mL in hydrothermal reaction kettle, add 22mL ultrapure water and be heated to 250 DEG C simultaneously
Stirring stops heating and naturally cools to room temperature, filters, take filtrate in bag filter (MWCO:1 kDa, pore after 10 hours
Size:ca. 1.0 nm) in be protected from light in the environment of ultrapure water dialysis for 24 hours, take supernatant liquor to be kept in dark place in 4 DEG C.
2. the synthesis of DMI
2,3,3- trimethyl -3H- indoles (4.8g, 30mmoL) and iodomethane (8.52g, 60mmoL) mixed dissolution are in 10mL acetonitrile
In, 60 DEG C of reflux 11h, naturally cool to room temperature under protection of argon gas, obtain a kind of lightpink precipitating, and ethyl acetate is used in filtering
It washes three times, vacuum drying obtains lightpink powder (8.7g, 96%)
(lightpink powder)；
Above-mentioned lightpink powder (0.150g, 0.50mmoL) and 4- diaminobenzene formaldehyde (0.112g, 0.75mmoL) is taken to mix molten
Solution is in 10mL ethyl alcohol, and 180 DEG C of reflux 4.5h, are evaporated under reduced pressure, remaining liq is through column Chromatographic purification (dichloromethane under protection of argon gas
Alkane: ethyl alcohol=22:1 volume ratio) it obtains pure small organic molecule (DMI), it reacts as follows:
；
3. the building of the fluorescence probe based on inner filtering effect: passing through ultraviolet-visible spectrum analysis, DMI has maximum suction at 546nm
Peak is received, simultaneously synthesizing CDs has maximum emission peak (λ at 520nmex=410nm), the two, which has, to be largely overlapped, i.e. DMI energy
The transmitting light for largely sponging CDs shows as CDs fluorescent quenching, and inner filtering effect occurs, successfully constructs fluorescence probe.
Embodiment 4
1. the synthesis of fluorescence nano carbon dots
Fluorescence nano carbon dots (CDs) are made by pyrolysis tomato, specific as follows: being weighed new fresh tomato 5.0g in mortar, filled
Divide after grinding through 150 mesh membrane filtrations, takes filtrate 3mL in hydrothermal reaction kettle, add 23mL ultrapure water and be heated to 250 DEG C simultaneously
Stirring stops heating and naturally cools to room temperature, filters, take filtrate in bag filter (MWCO:1 kDa, pore after 10 hours
Size:ca. 1.0 nm) in be protected from light in the environment of ultrapure water dialysis for 24 hours, take supernatant liquor to be kept in dark place in 4 DEG C.
2. the synthesis of DMI
2,3,3- trimethyl -3H- indoles (6.4g, 40mmoL) and iodomethane (11.36g, 80mmoL) mixed dissolution are in 10mL second
In nitrile, 60 DEG C of reflux 11h, naturally cool to room temperature under protection of argon gas, obtain a kind of lightpink precipitating, filtering, with acetic acid second
Ester is washed three times, and vacuum drying obtains lightpink powder (11.6g, 96%).
(lightpink powder)；
Above-mentioned lightpink powder (0.150g, 0.50mmoL) and 4- diaminobenzene formaldehyde (0.112g, 0.75mmoL) is taken to mix molten
Solution is in 10mL ethyl alcohol, and 180 DEG C of reflux 4.5h, are evaporated under reduced pressure, remaining liq is through column Chromatographic purification (dichloromethane under protection of argon gas
Alkane: ethyl alcohol=23:1 volume ratio) it obtains pure small organic molecule (DMI), it reacts as follows:
；
3. the building of the fluorescence probe based on inner filtering effect: passing through ultraviolet-visible spectrum analysis, DMI has maximum suction at 546nm
Peak is received, simultaneously synthesizing CDs has maximum emission peak (λ at 520nmex=410nm), the two, which has, to be largely overlapped, i.e. DMI energy
The transmitting light for largely sponging CDs shows as CDs fluorescent quenching, and inner filtering effect occurs, successfully constructs fluorescence probe.
Application examples
Fluorescence probe is to hydrogen sulfide imaging analysis (as shown in Figure 5) in living cells with the increase that concentration of hydrogen sulfide is added, cell
Interior green fluorescence intensity constantly enhances.
It is incubated for 24 hours Step 1: 80 μ g/mL fluorescence CDs are added in Tissue Culture Dish with living cells, it is slow with PBS
Fliud flushing washing cell removes the fluorescence CDs for not entering cell, is observed using laser confocal microscope system, cell is lasting
Issue green fluorescence.
It is incubated for 24 hours, uses with living cells Step 2: the mixed liquor of 100 μ L CDs and DMI is added in Tissue Culture Dish
PBS buffer solution washing cell removes the liquid for not entering cell, is observed using laser confocal microscope system, cell is green
Color less fluorescence, compared to step 1, its cell fluorescence intensity is substantially reduced.
Step 3: being separately added into concentration into the culture dish of step 2 is 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 200 μ
M, 500 μM, 1mM sodium sulfide solution incubation 1h, wash the liquid that cell removing does not enter cell with PBS buffer solution, using laser
Laser Scanning Confocal Microscope system is observed, and cell green fluorescence intensity about restores to the fluorescence intensity of step 1.
Implementation result example
Specific detection of the fluorescence probe to hydrogen sulfide
In the PBS buffer solution of pH=7.4, in the presence of 50 μm of oL/L sodium sulfide solutions, vulcanized sodium is to fluorescence
The response speed of probe is very fast, and under 410nm excitation wavelength, the fluorescence of probe is pulled up quickly, and in 7min, fluorescence is drawn
Play about 80%(Fig. 6), in subsequent 1 hour, fluorescence is held essentially constant.Should the result shows that, hydrogen sulfide pull-up probe it is glimmering
Light is very more and fast, implies that the fluorescence probe is quick, stable, sensitive.The fluorescence probe detect sulfurated hydrogen detection limit down to
0.42 μm of ol/L, linear response range: 10-70 μm of ol/L(R 2=0.9904).Simultaneously have detected probe to other interfere from
The response (Fig. 7) of son or molecule, it can be seen that probe is to Cl-、CH3COO-、SO4 2-、Br-、HCO3 -、I-、HClO、GSH、Hcy、
Cys、H2O2、K+、Na+、Ca2+Deng being almost not responding to, probe is shown to the specificity of hydrogen sulfide.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. detecting the fluorescent carbon point nano-probe of hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect, feature exists
In: fluorescent carbon point and small organic molecule receptor are based on inner filtering effect and form fluorescent carbon point nano-probe.
2. the fluorescent carbon point according to claim 1 for detecting hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect
Nano-probe, it is characterised in that: the fluorescent carbon point is prepared by tomato carbonization, and small organic molecule receptor chemistry formula is。
3. the fluorescent carbon point according to claim 2 for detecting hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect
Nano-probe, which is characterized in that the synthetic method of the fluorescent carbon point, steps are as follows: new fresh tomato is placed in mortar, is filled
Divide 100-260 mesh filter membrane excessively after grinding to obtain filtrate, take the filtrate of certain volume in hydrothermal reaction kettle, adds ultrapure water heating
It to 250 DEG C and stirs, stops heating cooled to room temperature after 10h, filtering is collected filtrate and is placed in bag filter, in ultrapure water
Dialysis is protected from light under environment for 24 hours, taking supernatant is fluorescent carbon point.
4. the fluorescent carbon point according to claim 3 for detecting hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect
Nano-probe, it is characterised in that: the volume ratio of the filtrate and ultrapure water is 3:(20-23)；Bag filter model MWCO:1
kDa, pore size: ca. 1.0 nm。
5. the fluorescent carbon point according to claim 2 for detecting hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect
Nano-probe, which is characterized in that the synthetic method of the small organic molecule receptor, steps are as follows:
(1) take 2,3,3- trimethyl -3H- indoles and iodomethane mixed dissolution in acetonitrile, under protection of argon gas 60 DEG C of reflux
11h naturally cools to room temperature, obtains a kind of lightpink precipitating, and filtering is washed three times with ethyl acetate, and vacuum drying obtains lightpink
Powder；
(2) the lightpink powder and 4- diaminobenzene formaldehyde mixed dissolution obtained step (1) is protected in 10mL ethyl alcohol in argon gas
Lower 180 DEG C of reflux 4.5h is protected, is evaporated under reduced pressure, remaining liq obtains pure small organic molecule receptor through column Chromatographic purification.
6. the fluorescent carbon point according to claim 5 for detecting hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect
Nano-probe, it is characterised in that: substance withdrawl syndrome of 2,3, the 3- trimethyl -3H- indoles in acetonitrile is in the step (1)
The substance withdrawl syndrome of 1-4mmol/mL, iodomethane in acetonitrile is 2-8 mmol/mL.
7. the fluorescent carbon point according to claim 5 for detecting hydrogen sulfide imaging in hydrogen sulfide and living cells based on inner filtering effect
Nano-probe, it is characterised in that: in the step (2), the substance withdrawl syndrome of lightpink powder in ethanol is 0.05mmol/
The substance withdrawl syndrome of mL, 4- diaminobenzene formaldehyde in ethanol is 0.075mmol/mL；The reagent that column Chromatographic purification uses for
Methylene chloride and ethyl alcohol is by volume (20-23): 1.
8. the application method of the described in any item fluorescent carbon point nano-probes of claim 1-7, which is characterized in that step are as follows: will
Fluorescent carbon point and small organic molecule receptor are incubated for 24 h with living cells in Tissue Culture Dish, wash cell with PBS buffer solution and remove
The liquid for removing not enter cell, is observed using laser confocal microscope system, it is dense to be then separately added into multiple groups gradient again
The sodium sulfide solution of degree is incubated for 1h, washs the liquid that cell removing does not enter cell with PBS buffer solution, aobvious using laser co-focusing
Micromirror systems are observed.
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| CN110903234A (en) * | 2019-11-07 | 2020-03-24 | 上海师范大学 | Hemicyanine fluorescent probe for detecting tabus metrorrhagia poison gas simulant DCNP, and synthetic method and application thereof |
| CN111100636A (en) * | 2019-12-25 | 2020-05-05 | 太原师范学院 | Styrene cyanine dye functionalized carbon dot sensor and preparation method and application thereof |
| CN114620709A (en) * | 2022-03-14 | 2022-06-14 | 哈尔滨理工大学 | Carbon dot-whole cell biological compound system for producing hydrogen |
| CN115181567A (en) * | 2022-07-11 | 2022-10-14 | 山西医科大学 | Detect H 2 FRET fluorescent probe of S, preparation method and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110903234A (en) * | 2019-11-07 | 2020-03-24 | 上海师范大学 | Hemicyanine fluorescent probe for detecting tabus metrorrhagia poison gas simulant DCNP, and synthetic method and application thereof |
| CN110903234B (en) * | 2019-11-07 | 2022-10-21 | 上海师范大学 | Hemicyanine fluorescent probe for detecting tabus metrorrhagia poison gas simulant DCNP, and synthetic method and application thereof |
| CN111100636A (en) * | 2019-12-25 | 2020-05-05 | 太原师范学院 | Styrene cyanine dye functionalized carbon dot sensor and preparation method and application thereof |
| CN111100636B (en) * | 2019-12-25 | 2022-06-24 | 太原师范学院 | Styrene cyanine dye functionalized carbon dot sensor and preparation method and application thereof |
| CN114620709A (en) * | 2022-03-14 | 2022-06-14 | 哈尔滨理工大学 | Carbon dot-whole cell biological compound system for producing hydrogen |
| CN114620709B (en) * | 2022-03-14 | 2023-10-03 | 哈尔滨理工大学 | Carbon dot-whole cell biological compound system for hydrogen production |
| CN115181567A (en) * | 2022-07-11 | 2022-10-14 | 山西医科大学 | Detect H 2 FRET fluorescent probe of S, preparation method and application |
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